The Hsp90s are a family of molecular chaperones that play important roles in regulating and maintaining the functionality of cells under proteotoxic stress and pathogenic pressure (Workman, P., Burrows, F., Neckers, L. & Rosen, N. Drugging the cancer chaperone Hsp90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann. N.Y. Acad. Sci. 1113, 202-216 (2007)). In humans, cytoplasmic heat shock protein 90 alpha and beta (Hsp90α and β), endoplasmic reticulum (ER) glucose-regulated protein 94 (Grp94) and the mitochondrial tumor necrosis factor receptor-associated protein 1 (Trap-1) are the four known Hsp90 paralogs (Sreedhar, A. S., Kalmar, E., Csermely, P. & Shen, Y. F. Hsp90 isoforms: functions, expression and clinical importance. FEBS letters. 562, 11-15 (2004); Johnson, J. L. Evolution and function of diverse Hsp90 homologs and cochaperone proteins. Biochim. Biophys. Acta. 1823, 607-613 (2012)). These proteins are ATP dependent and belong to the GHKL (Gyrase B, Hsp90, Histidine Kinases, MutL) ATPase superfamily, which is characterized by a distinct ATP binding “Bergerat fold” located in the N-terminal domain (NTD) (Chene, P. ATPases as drug targets: learning from their structure. Nat. Rev. Drug Discov. 1, 665-673 (2002)). Binding and release of the nucleotide drives the catalytic cycle of the Hsp90s and thereby assists in the refolding of client proteins through a series of association-dissociation catalytic cycles. Occupancy of this regulatory pocket by small molecule inhibitors inactivates Hsp90 chaperone function, and several pan-Hsp90 inhibitors have demonstrated potent reversal of the disease phenotype when tested in models of cancer, neurodegeneration, infection, and inflammatory disease. Due to these therapeutic activities, a select number of these compounds have also moved to the clinic for the treatment of cancers (Jhaveri, K., Taldone, T., Modi, S. & Chiosis, G. Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers. Biochim. Biophys. Acta. 1823, 742-755 (2012))
Despite considerable interest in the use of pharmacologic Hsp90 inhibitors for the treatment of disease, little is known about the contribution of each paralog to the observed therapeutic benefit. To date, all published studies have used pan-Hsp90 inhibitors to inactivate Hsp90s and the processes that depend on them, making it impossible to correlate the role of individual paralogs with the biological effects. This is particularly unsatisfying, considering that the chaperoning roles of these Hsp90s do not overlap. Thus, for example, while there is a considerable literature on the response of cytosolic Hsp90 to inhibitors, no study satisfactorily differentiates the role of the α and β paralogs. Furthermore, although both Grp94 and Trap-1 are abundant in the cancer cell, little is known about their contribution to the malignant phenotype (Sreedhar, A. S., Kalmar, E., Csermely, P. & Shen, Y. F. Hsp90 isoforms: functions, expression and clinical importance. FEBS letters. 562, 11-15 (2004); Johnson, J. L. Evolution and function of diverse Hsp90 homologs and cochaperone proteins. Biochim. Biophys. Acta. 1823, 607-613 (2012); Marzec, M., Eletto, D. & Argon, Y. GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim. Biophys. Acta. 1823, 774-787 (2012); Chen, B. The HSP90 family of genes in the human genome: Insights into their divergence and evolution. Genomics 86, 627-637 (2005)).
In large part the predicament of being unable to study individual paralogs in cancer cells, despite their divergent roles, stems from a lack of suitable tools. While pan-Hsp90 inhibitors, genetic manipulations in yeast and human cells, mutant cell lines, and gene deficient mice have shed light on several Hsp90-dependent cancer mechanisms, many challenges still remain. In particular, strategies that address the biology of Hsp90s and their individual paralogs in an endogenous cellular environment where the chaperones are limiting but not absent (i.e. in un-engineered cancer cell lines and in primary samples) are needed. Ideally, this gap can be filled by chemical tools that probe and manipulate a protein's function in a controlled manner. Such tools would complement traditional biochemical and biological approaches by aiding the molecular characterization of biomolecules both in vitro and within their natural biological contexts.
While useful both as therapeutics and as tools to dissect the cell-specific effects and mechanisms associated with Hsp90 paralogs in select phenotypes, the discovery of paralog specific Hsp90 inhibitors is particularly challenging because of a high degree of conservation in their ATP regulatory ligand binding cavities, the pocket to which the known synthetic ligands bind. Indeed, we and others found that most reported Hsp90 inhibitors bind equally well to the majority of these paralogs (Marzec, M., Eletto, D. & Argon, Y. GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim. Biophys. Acta. 1823, 774-787 (2012); Schulte, T. W. et al. Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones. Mol. Endo. 13, 1435-1448 (1999). Crystal structures of the cytoplasmic Hsp90 (α and β) N-terminal domain, either in the apo form or in complex with regulatory nucleotides or small molecules, are essentially superimposable (Immormino, R. M., Kang, Y., Chiosis, G. & Gewirth, D. T. Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors. J. Med. Chem. 49, 4953-4960 (2006); Soldano, K. L., Jivan, A., Nicchitta, C. V. & Gewirth, D. T. Structure of the N-terminal domain of GRP94: Basis for ligand specificity and regulation. J. Biol. Chem. 279, 48330-48338 (2003)). In addition, while slightly different docking orientations were observed for some small molecule ligands when bound to Hsp90 and Grp94, these have, as of yet, failed to translate into appreciable selectivity and specific cellular activity through individual paralog inhibition (Marzec, M., Eletto, D. & Argon, Y. GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum. Biochim. Biophys. Acta. 1823, 774-787 (2012); Immormino, R. M. et al. Different poses for ligand and chaperone in inhibitor-bound Hsp90 and GRP94: implications for paralog-specific drug design. J. Mol. Biol. 388, 10331042 (2009); (Duerfeldt, A. S., et al. Development of a Grp94 inhibitor. J. Am. Chem. Soc. 134, 9796-9804 (2012)).
Paradoxically, despite the high degree of sequence conservation in their ATP binding pockets, crystallographic and biochemical studies have shown that when bound to nucleotides, Hsp90α/β, Grp94 and Trap-1 adopt distinctly different conformations and hydrolyze ATP with notably different rates. Specifically, when bound to adenyl imidodiphosphate (AMP-PNP) a non-hydrolyzable ATP analog, the “lids” of the two N-terminal domains (NTD) of the yeast Hsp90α dimer move from the “open” to the “closed” conformation, trapping the bound nucleotide within the ATP binding cavity. The two closed NTDs then meet to form a second dimer interface that supplements the obligatory dimeric interactions contributed by the two C-terminal domains and importantly, aligns the catalytic residues for ATP hydrolysis. In contrast, the NTD “lids” of Grp94 do not close upon nucleotide binding but instead adopt a unique “extended open” conformation that does not cover the ATP binding pocket and does not allow for strong dimeric interactions between NTDs. As a result, nucleotide-bound Grp94 adopts a twisted “V” shape with their NTDs not symmetrically opposed, but, rather, oriented in opposite directions (Ali, M. M. et al. Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013-1017 (2006); Dollins, D. E., Immormino, R. M. & Gewirth, D. T. Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basis for nucleotide-induced conformational change. J. Biol. Chem. 280, 30438-30447 (2005)). In Trap-1, ATP binding leads to a predominantly closed conformation, albeit with kinetics slower than in the cytosolic Hsp90 (Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J. & Reinstein, J. The ATPase cycle of the mitochondrial Hsp90 analog Trapl. J. Biol. Chem. 283, 11677-11688 (2008)). Nonetheless this is insufficient to commit Trap-1 to nucleotide hydrolysis and is instead followed by re-opening of the chaperone conformation. Together, the biochemical evidence suggests that the overall structure and conformational flexibility of the proteins plays an important role in configuring the ATP-binding sites of these chaperones.
In the present disclosure, we take advantage of the conformational distinctions between the paralogs and use the chemical diversity imprinted into the purine-scaffold class to demonstrate that the identification of Hsp90 paralog-specific ligands is possible. We explain the source of paralog binding specificity using structural and modeling analyses. We then use several of the identified paralog specific inhibitors to provide novel insights into the tumor-specific chaperoning of a client protein by individual Hsp90s.